1. Field of the Invention
This invention relates to a method of screening monoclonal antibody-producing clones to identify those producing antibodies which recognize a desired carbohydrate determinant, such as CA 19-9. Such antibodies are useful in immunopurification, immunodiagnosis, and immunotherapy. By this method, we have identified antibodies suitable for immunodetection at physiological pH of antigens bearing the CA19-9 determinant.
2. Information Disclosure Statement
Tumor-associated antigens are antigens which are present in the serum and tissues of cancer patients. Many such antigens are also expressed in embryonic tissues, and, at low levels, in the tissue and serum of healthy individuals. Many of the tumor-associated antigens are glycoproteins, glycolipids, or mucopolysaccarides.
The portion of an antigen to which an antibody binds is called the antigenic determinant, or epitope. An antibody raised against a glycoprotein may recognize a protein epitope, a carbohydrate epitope, or an epitope formed by the junction of the protein and carbohydrate moieties. It is known that the terminal carbohydrate sequence of a glycolipid may also be found on a glycoprotein, thus, a glycolipid and a glycoprotein may bear the same carbohydrate epitope. See Rauvala and Finne, FEBS Lett., 97:1 (1979); McIlhiney, et al., Biochem. J., 227: 155 (1985).
Hakomori, "Monoclonal Antibodies Directed to Cell-Surface Carbohydrates", in Monoclonal Antibodies and Functional Cell Lines: Progress and Applications Ch. 4 (1984) discusses the development of various anti-carbohydrate monoclonal antibodies. He notes that there are various difficulties in producing these antibodies. According to Hakomori, carbohydrate moieties in glycoproteins are only weakly immunogenic. While the carbohydrate chains in some glycolipids are said to be strongly immunogenic, others reportedly were weakly immunogenic, or perhaps not immunogenic at all
An ideal antibody reagent would always bind all to all antigens of interest, and only to those antigens; in practice, a modest degree of cross-reactivity is tolerated, which varies from one application to the next.
Only about seven monosaccharides have been identified in the oligosaccharides of mammalian glycoproteins and glycosphingolipids. These are sialic acid (acetylneuraminic acid), N-acetyl-D-galactosamine (GalNAc), N-acetyl-D-glucosamine (GlcNAc), D-galactose (Gal), D-mannose (Man), D-glucose (Glu) and L-fucose (Fuc). Glycosphingolipids are composed of a base (e.g., sphingosine), a fatty acid, and carbohydrate. The base is linked to the fatty acid via an amide linkage, and this substructure is called ceramide. The ceramide is glycosidically linked to the carbohydrate. Gangliosides are sialic acid-containing glycosphingolipids. The carbohydrate moieties of GSLs are the immunodominant portion of the molecule. See Yogeeswaran, "Cell Surface Glycolipids and Glycoproteins in Malignant Transformation", 38 Advances in Cancer Research 289 (1983).
One of the first tumor-associated glycoproteins to be identified was carcinoembryonic antigen. See Hansen, U.S. Pat. No. 4,180,499. As many as nine to twelve Fab antibody fragments may bind simultaneously to this 180,000 dalton molecule. Egan, et al., Cancer, 40: 458 (1977). The majority of the antigenic sites for which antibodies are available are polypeptide regions, however, antibodies which recognize carbohydrate epitopes of CEA are known. See Nichols, et al., J. Immunol., 135: 1911 (1985). Many anti-CEA antibodies react with binding sites which are dependent on the tertiary structure of the protein. Rogers, Biochim. & Biphys. Acta, 695: 227 (1983).
The carbohydrate structures of several erythrocyte glycoprotein and glycolipid antigens are known to behave as tumor and differentiation markers. See Feizi, et al., Nature, 314: 53 (1985).
One family of red blood cell surface antigens is called the "Lewis group." The Lewis-a determinant has been characterized as a trisaccharide, Beta-Gal(1-3)-[alpha-Fuc(1-4)]-GlcNac. Lewis-a determinants are found as terminal sugar sequences on both glycosphingolipids and on glycoproteins. See Watkins, "Biochemistry and Genetics of the ABO, Lewis, and P Blood Group Systems," in 10 Adv. Human Genetics Ch. 1 (1980). Artificial Lewis-a determinant-bearing antigens have been prepared. Lemieux, U.S. Pat. No. 4,137,401. Assays for Lewis-a antigens are known. See Steplewski, U.S. Pat. No. 4,607,009.
Monoclonal antibody 19-9, produced by a hybridoma prepared from spleen cells of a mouse immunized with cells of the human colon carcinoma cell line SW1116, detects a serum antigen which appears associated with gastrointestinal and pancreatic cancers.
The antibody 19-9) has been deposited as ATCC HB 8059. Koprowski, U.S. Pat. No. 4,471,057. The epitope of this antibody is reportedly a carbohydrate with the sugar sequence NeuNAc-alpha(2-3)-Gal-beta(1-3)-[Fuc-alpha(1-4)]-GlcNac-beta-(1-3)-Gal. Magnani, et al., Cancer Research, 43: 5489-92 (1983). It should be noted that this "CA19-9" epitope includes what may be described as a sialylated Lewis-a with an additional galactose unit. For its biosynthesis, see Hansson and Zopf, J. Biol. Chem., 260: 9388 (1985). It is known that the binding of 19-9 to CRC cell line SW1116 is abolished by pretreatment of the cells with neuraminidase. U.S. Pat. No. 4,471,057. In SW1116 extracts, it apparently occurs as a ganglioside (with the structure sialosyl-Le.sup.a -beta(1-3)-Gal-beta(1-4)-Glc-betal-Ceramide), but in serum, it is expressed as a mucin.
Mucins are glycoproteins of high molecular weight and high carbohydrate content. They are known to be secreted by the seroviscous tissues found in the mouth, lungs, cervix and intestines. They are believed to provide a protective coating, shielding cells from osmotic and pH gradients and from physical trauma. A typical mucin has a molecular weight in excess of 500,000 daltons and a carbohydrate content of 60-80%. A typical mucin may possess as many as 200 oligosaccharide chains attached to a polypeptide backbone. Tumor-associated oligasaccarides on mucins include CA 19-9 (a.k.a. GICA), DuPan-2, CA 1 and YPAN 1. Neuraminidase, an enzyme that selectively cleaves sialic acid from oligosaccharides, alters the antigenic activity of many of these mucins. Tittenhouse, et al., Laboratory Medicine, 16: 556 (1985).
One problem with natural antibody-producing cells is that is difficult to maintain them indefinitely in culture. In 1975, Kohler and Milstein introduced a procedure for the continued production of monoclonal antibodies using hybrid cells (hybridomas). It entailed the fusion of spleen (antibody-producing) cells from an immunized animal with an immortal myeloma cell line in order to obtain immortalized antibody-producing cells. In order to obtain a monoclonal antibody which would recognize a colorectal cancer-associated antigen, Koprowski immunized a mouse with CRC cells. The hydridomas he eventually produced were screened for their ability to produce antibodies which bound the immunizing cells. (It is also possible to obtain cells lines which continue to produce antibodies by other techniques, such as optimization of culture conditions.)
Herlyn, et al., J. Immunol. Meth., 80: 107-116 (1985) describes a typical screening. Mice were immunized with intact tumor cells of various types, spent medium from tumor cell cultures, membranes of tumor cells, glycolipid extracts of tumor cells, and PEG precipitates from the sera of cancer patients. The hybridomas (12,818 in number) were first screened with the spent medium in which cultured normal and malignant cells had been grown. The more promising hybridomas (95) were then screened with the cells themselves. Finally, 40 clones were screened with cancer patients' sera. One antibody, CO 29.11, was selected for detailed study. Its ability to bind to isolated sialylated Lewis-a glycolipid and to Lewis-a was compared to that of the previously developed 19-9 antibody.
Herrero-Zabaleta, Bull. Cancer, 74: 387-396 (1987) reported the use of immunoprecipitated (with antibody 19-9) components of the fluid of a mucinous ovarian cyst for mouse immunization. Splenocytes from the immunized mouse and myeloma cells were fused to obtain hybridomas. The hybridomas were screened for the ability to produce a pattern of immunoperoxidase staining on paraffin sections of esophageal mucosae which was similar to that generated by 19-9. Only the supernatants of five of the 150 hybridoma wells showed reactivity with this material and only one had a pattern similar to that of 19-9. The antibody (121 SLE) was compared with 19-9 for its ability to react with ovarian mucinous cyst extracts. Both were reactive. In addition, samples of the cyst wall were fixed in ethanol, embedded in paraffin, and cut into sections. The sections were then deparaffinized and incubated with neuraminidase. Both antibodies were tested for their ability to bind to the neuraminidase-treated sections and neither exhibited significant reactivity. This is a very time-consuming procedure. Herrero-Zabaleta did not use neuraminidase-treated mucinous cyst fluids in his study, and the his unpurified cyst fluids were used only for characterization and not for screening.
Kortright, WO 87/01392 proposes improving the binding of a monoclonal antibody to a human carcinoma tumor antigen by removing sialic acid which apparently sterically hindered formation of the immuno-complex. Schauer, TIBS 357 (September 1985) discusses the dual role of sialic acid in both masking and accentuating antigenicity, and sets forth the structures of the natural sialic acids.
A heterogeneous immunoassay is one in which the antigen of interest is bound into an antigen-antibody complex, and this complex is physically separated from the remainder of the sample. There are two basic types of heterogeneous immunoassay. In a sandwich assay, the sample antigen is bound by both an immobilized antibody and a labeled antibody, thus forming a ternary complex. For this to be possible, the antigen must bear at least two epitopes sufficiently far apart to permit the antigen to be simultaneously bound by both antibodies. See David, U.S. Pat. No. 4,376,110. Sandwich assays are, unfortunately, prone to displaying a "high dose hook effect." Cragle, U.S. Pat. No. 4,595,661.
In heterogeneous competitive immunoassays, the sample antigen competes with a known quantity of kit antigen for the binding sites of a kit antibody. Either the kit antigen may be labeled and the kit antibody immobilized, or vice versa. Competitive inhibition assays are generally thought to have the disadvantage of a short dynamic standard curve range as compared with sandwiched assays. Moreover, since only one binding event is required to produce a signal, the assay may be less discriminating.
The commercial assays for CA19-9, marketed by Centocor, Abbott, Commissariat a L'Energie Atomique, Hoffman-LaRoche, Inc., Sorin Biomedica and FujiRebio, are all sandwich assays.
Delvillano, Jr., WO 84/00758 describes a forward sandwich immunoassay for CA 19-9. Delvillano states that with regard to detecting CA19-9 antigen with the 19-9 antibody, the preferred pH was 2.5-6.5, and especially 4.5. Table 4 shows the effect of buffer pH on CA 19-9 detection. It is generally known that pH may alter the binding affinity of an antibody, and it has been suggested that the pH of an incubation may be selected to increase specificity. Mosmann, et al., J. Immunol., 125: 1152 (1980). It is also known that acidic pH may dissociate existing antigen-antibody complexes in serum samples. Thomson, et al., PNAS (USA) 64: 161 (1969).
Koprowski, U.S. Pat. No. 4,471,057 did describe a competitive immunoassay for a "monosialoganglioside" bearing the epitope recognized by antibody 19-9 (ATCC HB 8059). A serum sample was incubated with this antibody, and the mixture was then brought into contact with a surface having attached thereto a monosialoganglioside antigen from a known human CRC cell. The assay was commercially impractical, as it included three overnight incubations, two washes, and an indirect signal system. Significantly, none of the aforementioned commercial assays for CA19-9 are competitive assays.
Adachi, U.S. Pat. No. 4,389,392 describes a method for determining the level of tumor-associated glycoprotein or glycolipid in a sample by incubating the sample with a lectin and measuring the amount of bound or unbound lectin. Pukel, U.S. Pat. No. 4,507,391 discloses an immunoassay for a G.sub.D3 ganglioside. Similarly, Irie, U.S. Pat. No. 4,557,931 discloses an assay for G.sub.M2 ganglioside.
No admission is made that any of the foregoing references constitute prior art or pertinent prior art, that the publications accurately reflect the actual experimental work of the authors, or that the dates of publication are exact.